APETALA2/ETHYLENE respnse factor and basic helix-loop-helix tobacco transcription factors cooperatively mediate jasmonate-elicited nicotine biosynthesis.

Transcription factors of the plant-specific apetala2/ethylene response factor (AP2/ERF) family control plant secondary metabolism, often as part of signalling cascades induced by jasmonate (JA) or other elicitors. Here, we functionally characterized the JA-inducible tobacco (Nicotiana tabacum) AP2/ERF factor ORC1, one of the members of the NIC2-locus ERFs that control nicotine biosynthesis and a close homologue of ORCA3, a transcriptional activator of alkaloid biosynthesis in Catharanthus roseus. ORC1 positively regulated the transcription of several structural genes coding for the enzymes involved in nicotine biosynthesis. Accordingly, overexpression of ORC1 was sufficient to stimulate alkaloid biosynthesis in tobacco plants and tree tobacco (Nicotiana glauca) root cultures. In contrast to ORCA3 in C. roseus, which needs only the GCC motif in the promoters of the alkaloid synthesis genes to induce their expression, ORC1 required the presence of both GCC-motif and G-box elements in the promoters of the tobacco nicotine biosynthesis genes for maximum transactivation. Correspondingly, combined application with the JA-inducible Nicotiana basic helix–loop–helix (bHLH) factors that bind the G-box element in these promoters enhanced ORC1 action. Conversely, overaccumulation of JAZ repressor proteins that block bHLH activity reduced ORC1 functionality. Finally, the activity of both ORC1 and bHLH proteins was post-translationally upregulated by a JA-modulated phosphorylation cascade, in which a specific mitogen-activated protein kinase kinase, JA-factor stimulating MAPKK1 (JAM1), was identified. This study highlights the complexity of the molecular machinery involved in the regulation of tobacco alkaloid biosynthesis and provides mechanistic insights about its transcriptional regulators.

In situ localization associates biologically active plant natriuretic peptide immuno-analogues with conductive tissue and stomata

Plant natriuretic peptide immuno-analogues (irPNP) have previously been shown to affect a number of biological processes including stomatal guard cell movements, ion fluxes and osmoticum-dependent water transport. Tissue printing and immunofluorescent labelling techniques have been used here to study the tissue and cellular localization of irPNP in ivy (Hedera helix L.) and potato (Solanum tuberosum L.). Polyclonal antibodies active against human atrial natriuretic peptide (anti-hANP) and antibodies against irPNP from potato (anti-StPNP) were used for immunolabelling. Tissue prints revealed that immunoreactants are concentrated in vascular tissues of leaves, petioles and stems. Phloem-associated cells, xylem cells and parenchymatic xylem cells showed the strongest immunoreaction. Immunofluorescent microscopy with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG supported this finding and, furthermore, revealed strong labelling to stomatal guard cells and the adjacent apoplastic space as well. Biologically active immunoreactants were also detected in xylem exudates of a soft South African perennial forest sage (Plectranthus ciliatus E. Mey ex Benth.) thus strengthening the evidence for a systemic role of the protein. In summary, in situ cellular localization is consistent with physiological responses elicited by irPNPs reported previously and is indicative of a systemic role in plant homeostasis.

Molecular recognition of DNA by rigid [n]-polynorbornane-derived bifunctional intercalators: synthesis and evaluation of their binding properties

We have exploited the concept of multivalency in the context of DNA recognition, using novel chemistry to synthesize a new type of bis-intercalator with unusual sequence-selectivity. Bis-intercalation has been observed previously, but design principles for de novo construction of such molecules are not known. Our compounds feature two aromatic moieties projecting from a rigid, polynorbornane-based scaffold. The length and character of the backbone as well as the identity of the intercalators were varied, resulting in mono- or divalent recognition of the double helix with varying affinity. Our lead compound proved to be a moderately sequence-selective bis-intercalator with an unwinding angle of 27 and a binding constant of about 8 M. 9-Aminoacridine rings were preferred over acridine carboxamides or naphthalimides, and a rigid [3]-polynorbornane scaffold was superior to a [5]-polynorbornane. The flexibility of the linker connecting the rings to the scaffold, although less influential, could affect the strength and character of the DNA binding.

Impaired expression of notch signaling genes in aged human skeletal muscle

Notch signaling is essential for myogenesis and the regenerative potential of skeletal muscle: however, its regulation in human muscle is yet to be fully characterized. Increased expression of Notch3, Jagged1. Hes1, and Hes6 gene transcripts were observed during differentiation of cultured human skeletal muscle cells. Furthermore, significantly lower expressions of Notch1, Jagged1, Numb, and Delta-like 1 were evident in muscle biopsies from older men (60-75 years old) compared to muscle from younger men (18-25 years old). Importantly, with supervised resistance exercise training, expression of Notch1 and Hes6 genes were increased and Delta-like 1 and Numb expression were decreased. The differences in Notch expression between the age groups were no longer evident following training. These results provide further evidence to support the role of Notch in the impaired regulation of muscle mass with age and suggest that some of the benefits provided by resistance training may be mediated through the Notch signaling pathway.

Bis(3-endo-camphoryl)phosphinic acid, a non-racemic helical supramolecular host with aquapores

Bis(3-endo-camphoryl)phosphinic acid (1) was prepared by the reaction of the lithium enolate of D-(+)-camphor and phosphorous trichloride followed by an oxidative work up. Compound 1 crystallizes from wet toluene as monohydrate 1·H2O, which was investigated by X-ray crystallography. Molecules of 1 are associated by strong hydrogen bonds giving rise to the formation of a supramolecular helix. The interior channel of the helix is filled by a one-dimensional (1D) string of water molecules that are also associated by hydrogen bonding. The 1D string adopts a twisted zigzag conformation. Although the hydrogen bond networks are not cross-linked both the screw of the helix and the twist of the 1D string of water molecules are left-handed (M) and controlled by the chiral camphoryl residues situated on the exterior of the helix. The overall supramolecular structure is strongly reminiscent of aquaporin-1, a significant membrane-channel protein responsible for the transport of water into the cells.

Electrostatic complex of didodecyldimethylammonium and DNA: Langmuir monolayer, Langmuir–Blodgett film and dye recognition at air/water interface

DNA–didodecyldimethylammonium (DNA–DDDA) electrostatic complex was prepared and characterized through Fourier transformation infrared (FT-IR), 1H NMR and circular dichroism (CD) spectroscopy. When the dye molecule aqueous solutions were used as the subphase, the interaction between three dye molecules, acridine orange (AO), ethidium bromide (EB) and 5,10,15,20-tetrakis(4-N-methylpyridyl)porphine tetra(p-toluenesulfonate) (TMPyP) and the complex at air/solution interface were investigated through the surface pressure–area (π–A) isotherms, Brewster angle microscopy and UV-Vis spectroscopy, respectively. Our investigation indicates that the interaction capabilities of the three dyes to DNA–DDDA complex are different and present an order of TMPyP>AO>EB. For the interaction forms, we believe that TMPyP intercalates into the double helix of DNA, and AO adsorbs onto the surface of the DNA. As for EB, the measured signal is too weak to give a definite interaction form in the present experiment.

Mutated MESP2 causes spondylocostal dysostosis in humans

Spondylocostal dysostosis (SCD) is a term given to a heterogeneous group of disorders characterized by abnormal vertebral segmentation (AVS). We have previously identified mutations in the Delta-like 3 (DLL3) gene as a major cause of autosomal recessive spondylocostal dysostosis. DLL3 encodes a ligand for the Notch receptor and, when mutated, defective somitogenesis occurs resulting in a consistent and distinctive pattern of AVS affecting the entire spine. From our study cohort of cases of AVS, we have identified individuals and families with abnormal segmentation of the entire spine but no mutations in DLL3, and, in some of these, linkage to the DLL3 locus at 19q13.1 has been excluded. Within this group, the radiological phenotype differs mildly from that of DLL3 mutation–positive SCD and is variable, suggesting further heterogeneity. Using a genomewide scanning strategy in one consanguineous family with two affected children, we demonstrated linkage to 15q21.3-15q26.1 and furthermore identified a 4-bp duplication mutation in the human MESP2 gene that codes for a basic helix-loop-helix transcription factor. No MESP2 mutations were found in a further 7 patients with related radiological phenotypes in whom abnormal segmentation affected all vertebrae, nor in a further 12 patients with diverse phenotypes.

'Forbidden' disulfides : their role as redox switches

Seminal studies by Richardson and Thornton defined the constraints imposed by protein structure on disulfide formation and flagged forbidden regions of primary or secondary structure seemingly incapable of forming disulfide bonds between resident cysteine pairs. With respect to secondary structure, disulfide bonds were not found between cysteine pairs: A. on adjacent beta-stands; B. in a single helix or strand; C. on non-adjacent strands of the same beta-sheet. In primary structure, disulfide bonds were not found between cysteine pairs: D. adjacent in the sequence. In the intervening years it has become apparent that all these forbidden regions are indeed occupied by disulfide-bonded cysteines, albeit rather strained ones. It has been observed that sources of strain in a protein structure, such as residues in forbidden regions of the Ramachandran plot and cis-peptide bonds, are found in functionally important regions of the protein and warrant further investigation. Like the Ramachandran plot, the earlier studies by Richardson and Thornton have identified a fundamental truth in protein stereochemistry: "forbidden" disulfides adopt strained conformations, but there is likely a functional reason for this. Emerging evidence supports a role for forbidden disulfides in redox-regulation of proteins.

Balsamin, a novel ribosome-inactivating protein from the seeds of Balsam apple Momordica balsamina

Plant seeds, a rich source of proteins, are considered important for their application as functional ingredients in a food system. A novel ribosome-inactivating protein (RIP), balsamin was purified from the seeds of Balsam apple, Momordica balsamina. Balsamin was purified by ion exchange chromatography on CM Sepharose and gel filtration on superdex-75. It has a molecular weight of 28 kDa as shown by SDS-PAGE analysis. Balsamin inhibits protein synthesis in a rabbit reticulocyte lysate-based cell free translation assay with an IC50 of 90.6 ng ml⁻¹. It has RNA N-glycosidase activity and releases a 400-base long fragment termed the Endo fragment from 28S rRNA in the same manner as does saporin-6 from Saponaria officinalis. The N-terminal sequence analysis of the first 12 amino acids of balsamin revealed that it shares 83% similarity with type I RIP α-MMC from Momordica charantia and 50% similarity with β-MMC (from Momordica charantia), bryodin I (from Bryonia dioica) and luffin a (from Luffa cylindrica). Balsamin was further characterized by mass spectrometry. CD spectroscopic studies indicate that secondary structure of balsamin contains helix (23.5%), β-strand (24.6%), turn (20%) and random coil (31.9%). Thus RIPs activity expressed in vegetables like Momordica sp. advocates its usage in diet.

Altered intracellular localization and valosin-containing protein (p97 VCP) interaction underlie ATP7A-related distal motor neuropathy

ATP7A is a P-type ATPase that regulates cellular copper homeostasis by activity at the trans-Golgi network (TGN) and plasma membrane (PM), with the location normally governed by intracellular copper concentration. Defects in ATP7A lead to Menkes disease or its milder variant, occipital horn syndrome or to a newly discovered condition, ATP7A-related distal motor neuropathy (DMN), for which the precise pathophysiology has been obscure. We investigated two ATP7A motor neuropathy mutations (T994I, P1386S) previously associated with abnormal intracellular trafficking. In the patients' fibroblasts, total internal reflection fluorescence microscopy indicated a shift in steady-state equilibrium of ATP7AT994I and ATP7AP1386S, with exaggerated PM localization. Transfection of Hek293T cells and NSC-34 motor neurons with the mutant alleles tagged with the Venus fluorescent protein also revealed excess PM localization. Endocytic retrieval of the mutant alleles from the PM to the TGN was impaired. Immunoprecipitation assays revealed an abnormal interaction between ATP7AT994I and p97/VCP, an ubiquitin-selective chaperone which is mutated in two autosomal dominant forms of motor neuron disease: amyotrophic lateral sclerosis and inclusion body myopathy with early-onset Paget disease and fronto-temporal dementia. Small-interfering RNA (SiRNA) knockdown of p97/VCP corrected ATP7AT994I mislocalization. Flow cytometry documented that non-permeabilized ATP7AP1386S fibroblasts bound a carboxyl-terminal ATP7A antibody, consistent with relocation of the ATP7A di-leucine endocytic retrieval signal to the extracellular surface and partially destabilized insertion of the eighth transmembrane helix. Our findings illuminate the mechanisms underlying ATP7A-related DMN and establish a link between p97/VCP and genetically distinct forms of motor neuron degeneration.

8-modified-2'-deoxyadenosine analogues induce delayed polymerization arrest during HIV-1 reverse transcription

The occurrence of resistant viruses to any of the anti-HIV-1 compounds used in the current therapies against AIDS underlies the urge for the development of new drug targets and/or new drugs acting through novel mechanisms. While all anti-HIV-1 nucleoside analogues in clinical use and in clinical trials rely on ribose modifications for activity, we designed nucleosides with a natural deoxyribose moiety and modifications of position 8 of the adenine base. Such modifications might induce a steric clash with helix αH in the thumb domain of the p66 subunit of HIV-1 RT at a distance from the catalytic site, causing delayed chain termination. Eleven new 2′-deoxyadenosine analogues modified on position 8 of the purine base were synthesized and tested in vitro and in cell-based assays. In this paper we demonstrate for the first time that chemical modifications on position 8 of 2′-deoxyadenosine induce delayed chain termination in vitro, and also inhibit DNA synthesis when incorporated in a DNA template strand. Furthermore, one of them had moderate anti-HIV-1 activity in cell-culture. Our results constitute a proof of concept indicating that modification on the base moiety of nucleosides can induce delayed polymerization arrest and inhibit HIV-1 replication.

Distinct disulfide isomers of μ-Conotoxins KIIIA and KIIIB block voltage-gated sodium channels

In the preparation of synthetic conotoxins containing multiple disulfide bonds, oxidative folding can produce numerous permutations of disulfide bond connectivities. Establishing the native disulfide connectivities thus presents a significant challenge when the venom-derived peptide is not available, as is increasingly the case when conotoxins are identified from cDNA sequences. Here, we investigate the disulfide connectivity of μ-conotoxin KIIIA, which was predicted originally to have a [C1–C9,C2–C15,C4–C16] disulfide pattern based on homology with closely related μ-conotoxins. The two major isomers of synthetic μ-KIIIA formed during oxidative folding were purified and their disulfide connectivities mapped by direct mass spectrometric collision-induced dissociation fragmentation of the disulfide-bonded polypeptides. Our results show that the major oxidative folding product adopts a [C1–C15,C2–C9,C4–C16] disulfide connectivity, while the minor product adopts a [C1–C16,C2–C9,C4–C15] connectivity. Both of these peptides were potent blockers of NaV1.2 (Kd values of 5 and 230 nM, respectively). The solution structure for μ-KIIIA based on nuclear magnetic resonance data was recalculated with the [C1–C15,C2–C9,C4–C16] disulfide pattern; its structure was very similar to the μ-KIIIA structure calculated with the incorrect [C1–C9,C2–C15,C4–C16] disulfide pattern, with an α-helix spanning residues 7–12. In addition, the major folding isomers of μ-KIIIB, an N-terminally extended isoform of μ-KIIIA identified from its cDNA sequence, were isolated. These folding products had the same disulfide connectivities as μ-KIIIA, and both blocked NaV1.2 (Kd values of 470 and 26 nM, respectively). Our results establish that the preferred disulfide pattern of synthetic μ-KIIIA and μ-KIIIB folded in vitro is 1–5/2–4/3–6 but that other disulfide isomers are also potent sodium channel blockers. These findings raise questions about the disulfide pattern(s) of μ-KIIIA in the venom of Conus kinoshitai; indeed, the presence of multiple disulfide isomers in the venom could provide a means of further expanding the snail’s repertoire of active peptides.

Structure of the analgesic μ-Conotoxin KIIIA and effects on the structure and function of disulfide deletion

μ-Conotoxin μ-KIIIA, from Conus kinoshitai, blocks mammalian neuronal voltage-gated sodium channels (VGSCs) and is a potent analgesic following systemic administration in mice. We have determined its solution structure using NMR spectroscopy. Key residues identified previously as being important for activity against VGSCs (Lys7, Trp8, Arg10, Asp11, His12, and Arg14) all reside on an α-helix with the exception of Arg14. To further probe structure−activity relationships of this toxin against VGSC subtypes, we have characterized the analogue μ-KIIIA[C1A,C9A], in which the Cys residues involved in one of the three disulfides in μ-KIIIA were replaced with Ala. Its structure is quite similar to that of μ-KIIIA, indicating that the Cys1−Cys9 disulfide bond could be removed without any significant distortion of the α-helix bearing the key residues. Consistent with this, μ-KIIIA[C1A,C9A] retained activity against VGSCs, with its rank order of potency being essentially the same as that of μ-KIIIA, namely, NaV1.2 > NaV1.4 > NaV1.7 ≥ NaV1.1 > NaV1.3 > NaV1.5. Kinetics of block were obtained for NaV1.2, NaV1.4, and NaV1.7, and in each case, both kon and koff values of μ-KIIIA[C1A,C9A] were larger than those of μ-KIIIA. Our results show that the key residues for VGSC binding lie mostly on an α-helix and that the first disulfide bond can be removed without significantly affecting the structure of this helix, although the modification accelerates the on and off rates of the peptide against all tested VGSC subtypes. These findings lay the groundwork for the design of minimized peptides and helical mimetics as novel analgesics.

Structural classification of proteins through amino acid sequence using interval type-2 fuzzy logic system

This paper introduces a new multi-output interval type-2 fuzzy logic system (MOIT2FLS) that is automatically constructed from unsupervised data clustering method and trained using heuristic genetic algorithm for a protein secondary structure classification. Three structure classes are distinguished including helix, strand (sheet) and coil which correspond to three outputs of the MOIT2FLS. Quantitative properties of amino acids are used to characterize the twenty amino acids rather than the widely used computationally expensive binary encoding scheme. Amino acid sequences are parsed into learnable patterns using a local moving window strategy. Three clustering tasks are performed using the adaptive vector quantization method to derive an equal number of initial rules for each type of secondary structure. Genetic algorithm is applied to optimally adjust parameters of the MOIT2FLS with the purpose of maximizing the Q3 measure. Comprehensive experimental results demonstrate the strong superiority of the proposed approach over the traditional methods including Chou-Fasman method, Garnier-Osguthorpe-Robson method, and artificial neural network models.

Drying and denaturation characteristics of α-lactalbumin, β-lactoglobulin, and bovine serum albumin in a convective drying process

Drying and denaturation kinetics of aqueous droplets of α-lactalbumin (α-lac), β-lactoglobulin (β-lg), and bovine serum albumin (BSA) were measured in a convective drying environment. Single droplets having an initial droplet diameter of 2 ± 0.1 mm and containing 10% (w/v) protein concentration were dried using conditioned air (65 and 80 °C, 2-3% RH, 0.5 m/s velocity) for 600 s. The denaturation of these proteins was measured by using reversed-phase HPLC. At the end of 600 s of drying 13.3 and 19.4% α-lac was found to be lost due to denaturation at 65 and 80 °C, respectively. Up to 31.0% of β-lg was found to be denatured, whereas BSA was not found to be significantly (p > 0.05) denatured in these drying conditions. The formation and strength of skin and the associated morphological features were found to be linked with the degree of denaturation of these proteins. The secondary structure of these proteins was significantly (p < 0.05) affected and altered by the drying stresses. The β-sheet and random coil contents were increased in α-lac by 6.5 and 4.0%, respectively, whereas the α-helix and β-turn contents decreased by 5.5 and 5.0%, respectively. The β-sheet and random coil contents in β-lg were increased by 7.5 and 2.0%, respectively, whereas the α-helix and β-turn contents decreased by 3.5 and 6.0%, respectively. In the case of BSA the β-sheet, α-helix, and random coil contents were found to increase, whereas the β-turn content decreased.